Effects of ASEA beverage intake on endurance performance in mice

Amy M. Knab, David C. Nieman, R. Andrew Shanely, Jennifer J. Zwetsloot, Lynn Cialdella-Kam, Mary Pat Meaney.
Human Performance Laboratory, Appalachian State University, North Carolina Research Campus, Kannapolis, NC
Background Results
ASEA is a saline-based beverage that undergoes a proprietary process and contains reactive
redox-signaling molecules. Table 1: Treadmill Endurance Protocol
Time Speed Figure 2: Endurance Run Time Figure 3: Rate of Muscle Glycogen Depletion Figure 4: Carnitine Palmitoyltransferase I
(min) (m/min) Details
31P NMR and EPR experiments utilizing spin trap molecules (DIPPMPO) were used to explore the 1 0 adjustment to treadmill 100

ug/mg protein/run time (min)

0.06
ASEA beverage for free radicals. An additional experiment using 31P NMR DIPPMPO with and
5 10 "warm up" * 1.5
2 12 80

Run Time (min)

without superoxide dismutase was conducted. Results supported the presence of stable peroxyl *

(arbitrary units)
2 14

CPT-1 Content
2 16 60 0.04 1.0
and/or superoxide radicals in ASEA. 2 18
2 20 40
Speeds between 20-24 correspond to roughly 80%
0.02 0.5
2 22 VO2max for mice
The theory of hormesis involves repeated exposure to a mild physical, chemical, or biological stress Mice will stay at this speed until they reach
20

resulting in increased resistance to subsequent exposures to otherwise harmful doses of the same 2- end 24 exhaustion (sit on shock grid for 5 full seconds) 0
0.00 0.0
Placebo ASEA
stressors. The exposures to mild stressors are thought to induce beneficial cellular responses Placebo ASEA

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Study Design

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Endurance Run Time (minutes)

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leading to increased whole organism resistance to the stress. Common examples of this beneficial

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Estimated rate of muscle glycogen

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ASEA Run group significantly

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depletion. ASEA Run group

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response include, exercise, ischemic preconditioning, and caloric restriction (Mattson, 2008). ASEA different than Placebo Run group

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significantly different than Placebo

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ASEA Treatment Placebo Treatment (p<0.001).

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may increase exercise performance through a hormesis effect, but this has not yet been established. (1-week) (1-week)
Run group (p=0.017).
CPT-I as measured by Western Blot
in muscle tissue. No significant
differences between groups (p>0.05).

PURPOSE: To determine if mice given ASEA have increased endurance treadmill run times
compared to placebo and investigate potential mechanisms. ASEA Sedentary
Placebo Run
Placebo Sedentary Figure 5: -Hydroxyacyl-CoA Dehydrogenase Figure 6: Phospho/pan-ACC
ASEA Run Group: Group: Group:
Group:
N=15 N=15 N=15
N=15

Dehydrogenase Activity
Treadmill Sedentary (normal Sedentary (normal

Phospho/pan-ACC Content
1.5
Methods cage activity only)
Treadmill
cage activity only)

-Hydroxyacyl-CoA
15 *#
*

(arbitrary units)

(arbitrary units)
Animals: Six-month old male specific pathogen-free C57BL/6 mice (n =60) were purchased from Jackson Laboratory. Mice
were randomly assigned to each of the four treatment groups (n = 15 each). Mice were group housed (3-4/cage) and provided standard Figure 1: NMR Spectra analysis of ASEA beverage
1.0
rodent chow and water ad libitum. All animal procedures were reviewed and approved by the North Carolina Research Campus 10
IACUC.

Treatment and Design: ASEA or placebo (same ingredients as ASEA beverage without undergoing the proprietary processing) 5 0.5
was administered via gavage once per day for 1-week. The average body mass of all the mice at the start of the study determined the
volume of ASEA used for the gavaging, but the volume did not exceed 0.3mL. Following the 1-week treatment period (7 days) mice
were euthanized and tissues harvested for further analysis of outcome measures. Mice from the endurance testing treatment groups 0
were oriented to the treadmill in the following fashion: During the three day period preceding the maximal endurance test, mice were 0.0

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oriented (trained) to the treadmill for 15 min/day. Speeds for the training days were 10 m/min, 15 m/min, and 18 m/min respectively.

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Then, on the final day of treatment mice underwent the maximal endurance capacity test on the treadmill (Table 1). For the treadmill

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orientation and endurance protocols, mice were run on a multi- lane rodent treadmill (Columbus Instruments, Columbus OH) equipped

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with a shock grid at the back. When the mouse could either no longer run (as assessed by sitting on the shock grid with all 4 paws off of

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the belt for more than 5 seconds), the mouse was removed from the shock grid immediately and placed back into the home cage. The

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-HAD enzyme activity was measured

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mice were monitored for recovery for a period of at least 20 minutes following the orientation bouts. Mice were euthanized within 30

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minutes of the final endurance test. in muscle tissue. No significant Phosphorylated ACC was normalized to
differences between sedentary groups ACC content. Both were measured via
Glycogen: Post-exercise and end point liver and muscle glycogen levels were assayed using the Glycogen Assay Kit (700480, or run groups (p>0.05).
Cayman Chemical Company, Ann Arbor MI). Rate of muscle glycogen usage was estimated for both ASEA Run and Placebo Run Western Blot analysis in muscle tissue.
groups. Example Calculation: Average muscle glycogen (Placebo Sedentary) Average muscle glycogen (Placebo Run) / Average * - Significantly different from Sedentary
Placebo Run Time. within same treatment (p=0.02)
31PNMR spectrum of a mixture of DIPPMPO and ASEA beverage. # - Significantly different than Placebo
Enzyme Assays: -hydroxyacyl-CoA dehydrogenase (-HAD) activities were determined in whole gastrocnemius homogenates Run (p=0.045)
using methods previously described (Laye, 2009). Briefly, powdered frozen muscle was homogenized in buffer containing HEPES, Na Green numbers are peak chemical shifts, red numbers are integral
pyrophosphate, Na+, EDTA, Triton, and protease and phosphatase inhibitors. CS activity was measured in homogenate incubated in values of corresponding peaks.
buffer containing oxaloacetate and dithiobis(2-nitrobenzoic acid) (DTNB). Acetyl-CoA was added to the buffer and CS activity was
determined by the appearance of reduced DTNB at a wavelength of 405nm. -HAD activity was measured in homogenate incubated in
buffer containing triethanolamine, EDTA, and nicotinamide adenine dinucleotide (NADH). Acetyl-CoA was added to the buffer and -
HAD activity was determined by the disappearance of NADH at a wavelength of 340nm. All assays were performed at 37C.
Conclusions
Western Blotting: Western blotting was performed as previously described (Laye et al. 2009). The following antibodies were When adjusted to run time, the estimated rate of muscle glycogen depletion was different between ASEA Run and Placebo Run groups.
used: Carnitine Palmitoyltransferase-1 (CPT1) (Santa Cruz Biotechnology, Santa Cruz, CA), Acetyl-CoA Carboxylase (ACC), and Skeletal muscle phosphorylated acetyl-CoA carboxylase (p-ACC) was significantly increased in ASEA Run compared to ASEA Sedentary (p=0.020) and Placebo Run groups (p=0.045). Fatty
phospho-ACC (Ser79) (Cell Signaling, Danvers, MA). Whole gastrocnemius homogenates were separated by SDS-PAGE, transferred to
polyvinylidene fluoride membranes. Membranes were exposed to the appropriate primary and secondary antibodies and bands were
acyl CoA transport (CPT1), and beta-oxidation (beta-HAD) were not different between ASEA Run and Placebo Run groups.
visualized by chemiluminescence (Pierce SuperSignal, Fisher Scientific, Rockford, IL). Band density was determined using a ASEA increased run time to exhaustion by 29% in mice, potentially through less inhibition of fatty acid oxidation via increased P-ACC, and muscle glycogen sparing (30%).
ChemiDoc XRTS Molecular Imager and Image Lab Software (BioRad, Hercules, CA). Phosphorylated-ACC (Ser79) protein was
normalized to total ACC protein. The data support increased endurance capacity and altered substrate utilization in mice after one week of ASEA intake. Further research is warranted to determine if these findings are due to
hormesis influences from the ASEA beverage.
Statistical Analyses: Two-way ANOVA was performed. Following a significant F-ratio, Student s t-test were performed to
determine differences between treatments. Significance was established at P < 0.05
Mark P. Mattson. Hormesis Defined. Ageing Res Rev. 2008 January; 7(1): 1-7 Funding, Reoxcyn Discoveries Group